The advent of severe acute respiratory syndrome (SARS) in 2003 poignantly demonstrated the urgency of establishing rapid, sensitive, specific, and inexpensive tools for differential laboratory diagnosis of infectious diseases. Through an unprecedented global collaborative effort, the causative agent was rapidly implicated and characterized, serologic and molecular assays for infection were developed, and the outbreak was contained. Despite these successes, however, the diagnosis of SARS still rests on clinical, epidemiological, and laboratory criteria.
On Oct. 22, 2003, the World Health Organization (WHO) SARS International Reference and Verification Laboratory Network met to review the status of laboratory diagnostics in acute severe pulmonary disease. Quality assurance testing indicated that false-positive SARS coronavirus (CoV) PCR results were infrequent in network laboratories. However, participants registered concern that current assays did not allow simultaneous detection of a wide range of pathogens that could aggravate disease and/or result in clinical presentations similar to SARS. The importance of extending rapid molecular assays to include other respiratory pathogens was reinforced by the reappearance of SARS in China, and by reports of a new, highly-virulent influenza virus strain in Vietnam.
To date, there is available only a limited repertoire of sensitive, specific diagnostic assays that allow surveillance and clinical management of SARS and other pathogen-associated diseases. However, these are often not ideal. For example, immunofluorescence and enzyme-linked immunosorbent assays (ELISA) inconsistently detect antibodies to SARS-CoV before day 10 or 20 after the onset of symptoms, respectively (WHO Multicentre Collaborative Network for Severe Acute Respiratory Syndrome (SARS) Diagnosis. A multicentre collaboration to investigate the cause of severe acute respiratory syndrome. Lancet, 361:1730-33, 2003; Li and Xu, Profile of specific antibodies to the SARS-associated coronavirus. N. Eng. J. Med., 349:5-6, 2003). Thus, although helpful in tracking the course of infection at the population level, these serologic tools have limited utility in detecting infection at early stages, when there may be potential to implement therapeutic interventions or measures (e.g., quarantine).
Contrastingly, assays based on polymerase chain reaction (PCR) have the potential to detect pathogen-associated infection at earlier time points. Indeed, methods for cloning nucleic acids of microbial pathogens directly from clinical specimens offer new opportunities to investigate microbial associations in diseases. The power of these methods lies not only in their sensitivity and speed, but also in their potential to succeed where methods for pathogen identification, through serology or cultivation, may fail because of an absence of specific reagents or because of fastidious requirements for agent replication. Various methods are currently employed for cultivation-independent characterization of infectious agents. These can be broadly segregated into methods based on direct analysis of microbial nucleic acid sequences, methods based on direct analysis of microbial protein sequences, immunological systems for microbe detection, and host-response profiling. Any comprehensive arsenal should include most, if not all, of these tools.
Multiplexing is an approach to nucleic-acid detection that uses several pooled nucleic-acid samples simultaneously, thereby greatly increasing detection speed. In current multiplex PCR systems, the use of consensus primers reduces sensitivity because: (1) binding sites are not optimized; (2) optimal primers within a consensus pool are not present at optimal concentration; and/or (3) short regions that detect all organisms within a given taxon cannot be defined. Furthermore, conventional multiplex PCR assays do not allow sensitive detection of more than 10 genetic targets. Gel-based systems are cumbersome, and are limited to visual distinction of products that differ by 20 bp. Thus, multiplexing is restricted to the number of products that can be distinguished at 20-bp intervals within the range of 100-250 bp (where amplification efficiency decreases with larger products); nesting or Southern hybridization is generally required to achieve high sensitivity.
At present, the most sensitive of all multiplex assays is real-time PCR. Real-time PCR methods have significantly changed diagnostic molecular microbiology by providing rapid, sensitive, specific tools for detecting and quantifying genetic targets. Because closed systems are employed, real-time PCR is less likely than nested PCR to be disrupted by assay contamination arising from inadvertent aerosol introduction of amplicon/positive-control/cDNA templates that can accumulate in diagnostic laboratories. Real-time PCR is also very specific. This specificity, however, is both its strength and its weakness: although the potential for false-positive signals is low, so is the utility of the method for screening to detect related, but not identical, genetic targets.
Specificity in real-time PCR is provided by two primers (each approximately 20 matching nucleotides (nt) in length), combined with a specific reporter probe of about 27 nt. The constraints of achieving hybridization at all three sites may confound detection of diverse, rapidly-evolving microbial genomes, such as those of single-stranded RNA viruses. These constraints can be compensated for, in part, by increasing numbers of primer sets accommodating various templates. However, because real-time PCR relies on fluorescent reporter dyes, the capacity for multiplexing is limited to the number of emission peaks that can be unequivocally separated. At present, up to four dyes can be identified simultaneously. Although the repertoire may increase, it is unlikely to change substantively.
In view of the foregoing, a need still exists for enhanced multiplex capacity in diagnostic molecular microbiology, including enhanced capacity to detect coinfection. As specific antiviral therapies are established, early diagnosis of infection and coinfection will become increasingly important in minimizing morbidity and mortality resulting from infectious pathogens.